Re: Gram Stain

From:Paul Bradbury

Hi. I think the method you are talking about is the Brown-Hopp's variant of 
the Gram stain. We use this method regularly to demonstrate H. pylori in 
gastric biopsies.

After doing a more-or-less conventional Gram stain, the section is treated 
- acetone (to dehydrate it)
- acetone-picric acid (to stain the background yellow)
- acetone/xylene (to "wash off" excess picric acid, and begin clearing)
- xylene (to complete clearing prior to mounting)

Why do you want to get rid of the acetone-xylene step?

If you take the section from acetone-picric acid straight into pure xylene, 
any residual picric acid may precipitate out (picric acid is essentially 
insoluble in xylene).

It will probably be quite okay to take sections from acetone-picric acid 
into a "wash" with pure acetone (just to remove any excess picric acid). 
This will also complete the dehydration. Then treat with a couple of changes 
of xylene and mount.

Paul Bradbury, FIMLS, ART
Kamloops, BC Canada

----Original Message Follows----
Subject: Re: Gram Stain
Date: Fri, 25 Oct 2002 15:53:43 -0400 (EDT)

Dear John
After you use crystal violet, Gram Iodine  and counterstain, towards the end
of the stain you have to use a 50% solution of xylene and acetone. Then take
it into the xylene and coverslip. There is a step where Bouins and Formalin
mixture is used.
I am trying to get rid of the xylene/acetone step. I don't have the 
at home with me. Its just a stain that the lab uses where I work.
Thank you
sandi miller HT

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